DK146937B - TRIPEPTIDES OR SALTS THEREOF USED AS DIAGNOSTIC CHROMOGENT SUBSTRATE FOR SERINE PROTEASES - Google Patents

Description

(19) DENMARK Æ φ (12) PUBLICATION NOTICE 146937 Β

DIRECTORATE OF

THE PATENT AND TRADEMARKET SYSTEM

(21) Patent Application No: 3127/76 (51) lnt.CI.3: C 07 C103 / 52 .... .... . Λή. . _ 012 Q 1/34 (22) Date of filing: 09 Jul 1976 (41) Alm. available: 12 Jan 1977 (44) Submitted: 20 Feb 1984 (86) International Application No: - (30) Priority: 11 Jul 1975 SE7507974 (71) Applicant: SHARE COMPANY * KABI; S-104 25 Stockholm, SE.

(72) Inventor: Bo * Thureeson of Ekenstam; SEE, LeH Erik * Aurell; SE, Karl Goeran 'Claeson; SE, Birgitta Gunilla 'Karlsson; SE, Stig Ingemar 'Gustavsson; SE, Gun Anita 'Olausson; SEE.

(74) Plenipotentiary: Lehmann & Ree_ Engineering Company (54) Tripeptides or salts thereof for use as diagnostic chromogenic substrate for serine proteins

The present invention relates to tripeptides or salts thereof for use as diagnostic chromogenic substrate with good solubility and high specificity for serine proteases.

The substrates of the invention are particularly useful for quantitatively determining the aforementioned serine proteases classified as (EC 3.4.21) and cleaving the peptide chain on the carboxyl ® side of arginine or lysine. Furthermore, the substrates can be used

I for the study of reactions in which said enzymes are formed, inhibited Λ or degraded, or for the study of factors that influence or participate in such a reaction. Synthetic substrates for enzyme determination have great advantages over the natural ones, provided they meet certain conditions, such as high sensitivity and specificity to the enzyme, good solubility in water or in the biological test solution, and easy detectability of some of the cleavage products.

Substrates which are excellent for determining e.g. plasmin, thrombin, trypsin, kallikrein and urokinase are among others. disclosed in the specification of Swedish Patent No. 380,258 and are in principle chromogenic tripeptide derivatives. Among the best substrates of this type are those having a benzoylated N-terminal end and a chromophore group coupled to the C-terminal end, for example: 12 3

Benzoyl - A - A - A - p-nitroanilide I 12 3 wherein A, A and A are amino acids.

With specific amino acid sequences, it is possible to find among those substrates those which have a particular sensitivity to a certain or certain enzymes. During enzymatic hydrolysis, the substrates form the chromophore product p-nitroaniline, which can be readily detected spectrophotometrically. However, the substrates have a limitation due to their relatively poor solubility (less than or possibly equal to 1 mg / ml). A low solubility necessitates working very close to the saturation limit of the substrate to obtain a satisfactory substrate concentration. Therefore, by enzyme determinations in different biological systems, it may happen that either the substrate itself or a protein / substrate combination is precipitated. The precipitates mentioned cause misleading spectrophotometric readings and thus erroneous enzyme determinations.

Furthermore, from the above-mentioned Swedish patent no. 380,258, it is known to use tripeptides of the formula Leu-Leu-Arg-pNA and β-cyclohexy1-Ala-Val-Arg-pNA, wherein the terminal leucine and β-cyclohexylalanine, respectively, have L-form which diagnostic substrate for serine proteases. Thus, by replacing the N-terminal benzoyl group of the above Formula I with a hydrogen atom, enzyme substrates which are significantly more soluble are obtained. The free protonated amino group of amino acid A 1 increases solubility, but also causes the rate at which the enzyme cleaves the substrates to decrease (cf. Table II). Furthermore, in a biological test solution, the substrates can now be degraded in an undesirable manner from the N-terminal end of aminopeptidases.

According to the present invention, it has now surprisingly been found that in tripeptides of the general formula:

Η - A - A - A - pNA

1 2 where A and A are selected from the amino acids Gly, Ala, Val, Leu, Ile, 2 3

Pip, Pro, Aze, and A may further be Phe, and A is selected from amino acids Arg and Lys, replacing the L-form of amino acid A ^ with the D-form, obtaining enzyme substrates which, on the one hand, are very easily soluble but which at the same time has a much higher activity as a substrate for serine proteases than the corresponding tripeptides, wherein the amino acid A 1 has the L-form, yes often even higher than the corresponding benzoylated tripeptides. Furthermore, the N-terminal free D-amino acid in the novel tripeptides also prevents an undesired attack of amino-peptidases, as these are specific for L-amino acids.

The tripeptides according to the invention are therefore characterized in that they have the following general formula: H - D - A1 - A2 - A3 - NH -no2, 1 2 where A and A are selected from the amino acids Gly, Ala, Val, Leu, Ile, 2

Pip, Pro, Aze, and A may further be Phe, and A is selected from the amino acids Arg and Lys.

In the syntheses of the novel tripeptides of the invention, commonly known protecting groups and coupling methods are used, all of which are well known in the peptide chemistry.

As the α-amino protecting group, it is advantageous to use carbobenzoxy or t-butyloxycarbonyl or some related groups such as e.g. p-methoxy-, p-nitro- or p-methoxy-phenylazo-carbobenzoxy.

It is advantageous to use protonization or the NO2 or p-toluenesulfonyl groups to protect the 6-guanido group in the arginyl group.

For the protection of the ε-amino group in lysine, it is advantageous to use the carbobenzoxy, t-butyloxycarbonyl or p-toluenesulfonyl groups first and foremost.

As the leaving group of the α-carboxyl protecting group it is appropriate to use the methyl, ethyl or benzyl ester group.

The coupling between two amino acids or a dipeptide and an amino acid is achieved through activation of the α-carboxy group. The activated derivative may be either isolated or formed in situ and may e.g. be p-nitrophenyl, trichlorophenyl, pentachlorophenyl, N-hydroxy succinic acid or N-hydroxybenzotriazole ester, symmetric or asymmetric anhydride or acid azide.

4 146937

The activation of the above-mentioned ester derivatives is advantageously achieved by the presence of a carbodiimide e.g. Ν, Ν'-di-cyclohexylcarbodiimide, which can also serve as an activating coupling agent directly between the carboxy and amine components.

The principle of the peptide syntheses is the stepwise addition of the amino acids to the C-terminal arginyl group, which is either initially provided with a coupled chromophore group, which then acts as a carboxy protecting group, or is provided with a leaving carboxy protecting group, and the chromophore group is then coupled to the tripeptide derivative, or alternatively it is in principle possible to choose to synthesize the N-terminal dipeptide fragment itself, which is later coupled to the arginyl group with or without a chromophore group in principle as discussed above.

Regardless of the method chosen, a purification of the intermediate and end products by gel filtration chromatography is desirable as this method allows for rapid synthesis and provides maximum yields.

The invention will be described in more detail in the following non-limiting specific examples.

abbreviations

Amino acids (unless otherwise stated is meant the L-form):

Arg = arginine

Aze a = 2-Acetidine carboxylic acid

Ala = Alanine

Gly = Glycine

Ile = isoleucine

Leu = Leucine

List as Lysin

Phe s * Phenylalanine

Pip = Pipecolinic acid

Pro as Prolin

Selected = Selected

AcOH = Acetic Acid

Bz = Benzoyl

Cbo- = Carbobenzoxy-DCCI = Dicyclohexylcarbodiimide DMF = Dimethylformamide 5 146937

EtgN = Triethylamine

EtOAc = Ethyl acetate GPC = Gel filtration chromatography HBT = N-hydroxybenzotriazole HMPTA = Ν, Ν, Ν ', N', N'1, N '' - hexamethylphosphoric triamide HONSu = N-hydroxyacetic acid imide

MeOH = Methanol -OpNP = p-nitrophenoxy-pNA = p-nitroanilide tBoc = t-butyloxycarbonyl TFA = trifluoroacetic acid TLC = thin layer chromatography

Reaction types used in the syntheses.

In synthesizing the new tripeptides summarized in Table II, the different reaction steps are carried out in much the same way. For this reason, a general description of the different reaction types and the page in Table I gives an overview of intermediate and end products in reprocessing methods used in the different reaction types and certain physical data.

5®53S £ i22§ £ YE®_ii

Coupling of the chromophore group 20 mmol Na, N 2 -protected arginine or Na, N 2 -protected lysine or a similarly suitably protected finely divided and well-dried peptide derivative is dissolved in 50 ml of freshly distilled HMPTA at room temperature, then 20 mmol Et 2 N and 30 mmol of p-nitroaniline in the form of its isocyanate derivative is added under anhydrous conditions and with stirring. After 1 day reaction time, the reaction mixture is poured into 0.5 1 2% sodium bicarbonate solution with stirring. The resulting precipitate is removed by filtration and washed well with bicarbonate solution, water, 0.5 N hydrochloric acid and again water. From the precipitate, the desired product is extracted with e.g. methanol, certain by-products are not dissolved. After evaporation, the methanol extract can be crystallized from a suitable medium or purified by GPC.

6 146937

Reaction fabric_2 Λ

Pre-cleavage of a carbobenzoxy protecting group (Cbo-).

10 mmol of the well-dried Cbo derivative is slurried in 25 ml of dry AcOH and 15 ml of 5.6 N HBr in AcOH are added under anhydrous conditions at room temperature. After a reaction time of 45-6 min. the solution is dropped dropwise to 300 ml of dry ether with vigorous stirring. The ether phase is sucked from the formed precipitate, which is washed with 2-3 portions of ether per day. 100 ml. The hydrobromide thus formed of the Na-unblocked compound is dried over NaOH tablets in vacuo at 40 ° C for 3-16 hours.

Cleavage of a t-butyloxycarbonyl protecting group (tBoc-).

10 mmol of the well-dried tBoc derivative is dissolved in 200 ml of 25% TFA in CH 2 Cl 2 under anhydrous conditions at room temperature. After a reaction time of 20 minutes, the solution is added dropwise with 500 ml of dry ether. The formed precipitate is removed by filtration and washed extensively with ether. The trifluoroacetate thus formed of the Na-deblocked compound is dried over NaOH tablets in vacuo at 30 ° C for 2-3 hours.

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Coupling reactions.

Release of the α-amino group.

When acylating the formed derivatives by reaction type .2 or 3, the α-amino group must be present as a free base. The release can be accomplished in many different ways. Among other things, it is possible to add an equivalent of a dry tertiary amine (e.g.

A N or N-ethylmorpholine) to a DMF solution cooled to -10 ° C by the HBr or TFA derivative. In cases comprising Et 2 N and HBr derivatives, the precipitated Et 3 N * HBr is removed by filtration. Alternatively, the HBr or TFA derivative can be dissolved in a 5% sodium bicarbonate solution from which the released derivative is extracted with e.g. EtOAc or butanol, after which the organic phase is dried and evaporated.

7 146937 a) With Να protected active ester derivative.

To a solution of 10 mmol of peptide or amino acid derivative released according to the above in 20-50 ml of freshly distilled DMF at -10 ° C is added 11 mmol of Na-protected p-nitrophenyl or N-hydroxy succinic acid ester derivative of the amino acid to be switch on. After a reaction time of 1 hour at -10 ° C, the solution is buffered with 5 mmol of tertiary amine and then allowed to slowly adjust to room temperature. The course of the reaction is suitably followed by TLC analyzes.

If necessary, add another 5 mmol of base after a new cooling. When the reaction is complete, the solution is evaporated on a rotary evaporator to an oily residue which is stirred with a few portions of water. The residue is purified using GPC or recrystallization. When GPC is used to purify the coupling product, and this has an elution volume that is wholly or partially consistent with the elution volume of the active ester derivative of the coupled amino acid, contamination of the coupling product can be avoided if unused ester derivative after end reaction is replaced by an evaporation ( 3-5 mmol) of a primary amine e.g. n-butyl-amine for 30 minutes at room temperature. Then the work-up is done as described above.

b) With Na protected amino acid or peptide and formation of active ester in situ.

To a solution of 10 mmol of the aforementioned released peptide or amino acid derivative in 20-50 ml of freshly distilled DMF is added 11 mmol of Na-protected amino acid or similarly protected peptide derivative with a C-terminal free carboxy group, 11 mmol of LGBT or HONSu and 11 mmol DCCI at -10 ° C. After 1-3 hours at -10 ° C, the reaction solution is allowed to adjust to room temperature. The course of the reaction is suitably followed by TLC analyzes. After completion of the reaction, the solution is added with stirring to 100-300 ml of 5% NaHCO3 (aqueous).

The precipitate formed is washed with water after filtration or decantation. The residue is purified by GBP or by recrystallization.

8 146937 BSSiStionstyge ^^

Cleavage of all protecting groups, purification and ion exchange.

In 0.2-1.2 mmol of the protected peptide derivative containing the chromophore, the protecting groups are cleaved off by 5-20 ml of dry HF in the presence of 0.2 - 1.0 ml of anisole in an apparatus according to Sakakibara suitable for this purpose. 60 minutes at 0 ° C. After completion of the reaction and after all the HF is distilled off, the crude product is dissolved in 33% aqueous AcOH and purified by GPC. The product is isolated by freeze-drying from dilute AcOH and conducted for ion exchange on a column (containing a weak basic ion exchange resin Sephadex 'QAE-25 in chloride form swollen in MeOH: water, 95: 5 with the same medium as solution and The pure product is freeze-dried from water.

Gel filtration chromatography.

By using GPC on protected peptide or amino acid derivatives, crude products or evaporated mother liquors after crystallization, a simplified work-up procedure and optimal yields are obtained. The material is then dissolved in MeOH and transferred to a column of appropriate size (the 0.5-7.5 l, 100 cm length) (R) packed with Sephadex LH-20, which is swollen in MeOH, and eluted with the same solvent. The eluate is fractionated with appropriate partial volumes and its UV absorption (254 nm) is continuously investigated. Product-containing gel fractions are examined for purity by TLC and the pure collected and evaporated.

To purify the peptide derivatives after cleavage of the protecting groups by HF according to 5 above, the 30% aqueous AcOH solution of the crude product is transferred to a column of appropriate size (volume 0.5-2.0 L, length 60 cm) packed with Sephadex G-15, which is swollen in 30% aqueous AcOH and eluted with the same solvent. Following the process of the above, the product-containing pure part fractions are freeze-dried if desired after partial evaporation on rotary evaporator at 25 ° C.

Thin layer chromatography.

For the TLC analysis, glass plates prepared with "Silica Gel ^ 254" (Merck) are used as absorbent. The solution systems used (volume ratio) are: 9 146937 A: n-butanol: AcOH's water (3: 2: 1) p · *: chloroform: MeOH (9: 1) P chloroform: MeOH (19: 1)

After chromatography, the plate is studied in UV light (254 run) and developed with Cl / o-toluidine reagent according to common practice. The indicated R 2 values are the results of separate chromatographs.

Determination of serine proteases with chromogenic substrates.

The substrates prepared according to the following examples are used to determine various enzymes according to the process outlined below. The principle of the assay is based on the fact that the cleavage product formed by enzymatic hydrolysis has a UV spectrum that is substantially different from the substrate. Thus, all the p-nitroanilide substrates of the invention have absorption maxima of about 300 nm with a molar extinction coefficient of about 12,000. At 405 nm, the absorption of these substrates is almost stopped. p-Nitroaniline which is cleaved from the substrate during enzymatic hydrolysis has an absorption maximum of 380 nm and a molar extinction coefficient of 13,200, which at 405 nm has only decreased to 9620. Thus, by spectrophotometric determinations at 405 nm it is easy to follow the amount of formed p-nitroaniline, which is proportional to the degree of enzymatic hydrolysis, which in turn is determined by the active amount of enzyme. Table II shows a comparison of relative reaction rates of previously known substrates of formula I, their non-benzoylated forms and substrates of the invention. This table clearly shows the advantages of the substrates according to the invention.

Thus, it is seen that the substrates according to the invention are several times better than corresponding substrates with N-terminal La-minoic acid and furthermore at least as good as the previously known best substrates as of the benzoylated substrates of formula I. Furthermore, the greater solubility of the new substrates (about 20-300 times greater) a very big advantage of enzyme determinations, especially in biological systems, in which the poorer solubility of previously known substrates caused problems partly due to the fact that substrate saturation could not be achieved and partly because of the risk of unwanted precipitation.

146937 ίο (R)

The gel filtration gel Sephadex G-15 is a (R) cross-linked dextrangel. The gel Sephadex LH-20 is a hydroxypropy- (R) pelleted crosslinked dextrangel. The ion exchanger used Sephadex's QAE-25 is a cross-linked dextran with diethyl- (2-hydroxypropyl) -aminoethyl as a functional group. The gels are from Pharmacia Fine Chemicals, Upsala, Sweden.

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TABLE II

Substrate Solubility, Relative Reaction Rate mg / ml Bufferx / "T-T ^ -ΡΪ-Kil -OK ~

Bz-Val-Pro-Arg-pNA 0.3 6 60 15 H-Val-Pro-Arg-pNA 40 6 55 15 H-D-Val-Pro-Arg-pNA (XIII) 100 80 100 95

Bz-Val-Pip-Arg-pNA 4 45 30 H-Val-Pip-Arg-pNA 4 35 45 H-D-Val-Pip-Arg-pNA (XIV) 100 70 100

Bz-Val-Leu-Arg-pNA 0.2 100 H-Val-Leu-Arg-pNA 50 H-D-Val-Leu-Arg-pNA (XVI) 600 100

Bz-Val-Leu-Lys-pNA 0.5 100 H-Val-Leu-Lys-pNA 25 H-D-Val-Leu-Lys-pNA (XVIII)> 100 100

Bz-Ile-Leu-Arg-pNA 0.2 55 100 H-Ile-Leu-Arg-pNA 10 H-D-Ile-Leu-Arg-pNA (XV) 4 75 100

Bz-Ile-Leu-Lys-pNA 1 130 H-Ile-Leu-Lys-pNA 35 HD-Ile-Leu-Lys-pNA (XVII) 20 x / buffer = Tris, pH = 8.2, ionic strength = 0 In the table above, the relative reaction rates of the various substrates are given in relation to a reference substrate selected for each enzyme. Symbols, reference substrates and their sensitivity to the respective enzymes are as follows:

Enzyme (Symbol) Reference Substrate No. Sensitivity (indicated amount of enzyme giving activity 0/1 nkat) AOD / min = 0.0254

Thrombin (T) XIV 0.06 NIH

Trypsin (Try) XIII 0.03 µg (Novo)

Plasmin (PI) XVIII 0.01 CU

Kallikrein (Kai) XVI 0.2 BE

Urokinase (UK) XIV 40 pe

Plasma chalice (hp) XL 0.002 PEU

Where: AOD = change of optical density (absorbance) nkat = nanocatal; 1 catal is the amount of enzyme which converts 1 mole of substrate per second

NIH = National Institute of Health, which has determined the thrombin unit NIH

CU = Casein Unit (Casein Unit) BE = Bayer Einheit (Bayer Unit) PE = PU = Plow Unit (Plow Unit) PEU = Plasma Equivalent Unit (i.e., as much as found in 1 ml of activated normal plasma).

17 146937

Additional examples for TABLE II

Substrate Relative reaction rate __Enzyme___T Try PI Kai Pk

Ref. Substrate (Act. = 100) XIV XIII XVIII XVI XL

H-Ala-Ala-Arg-pNA 4 10 2 HD-Ala-Ala-Arg-pNA (XXXV) 100 50 15 a-Leu-Gly-Arg-pNA 4 4 HD-Leu-Gly-Arg-pNA (XXXVI) 60 15 H-Val-Aze-Arg-pNA 10 70 HD-Val-Aze-Arg-pNA (XXXIX) 105 155 H-Pro-Phe-Arg-pNA 10 80 60 25 HD-Pro-Phe-Arg-pNA ( XL) 20 130 90 100 H-Pip-Phe-Arg-pNA 15 60 60 20 HD-Pip-Phe-Arg-pNA (XLI) 30 130 120 75 H-Pro-Phe-Lys-pNA 40 HD-Pro-Phe -Lys pNA (XLII) 130

Enzyme designation; T = Thrombin

Try = Trypsin

Pi = Plasmin

Kai = Kallikrein

Pk = plasma plasma act. = activity (reaction rate)